Secreted antigens of Mycobacterium tuberculosis (Mtb) induce strong T cell responses and interferon-gamma (IFN-γ) secretion, both of which are integral in the defense against Mtb. We used web-based tools (SignaIP and Prosite) to identify putative secreted proteins from Mtb genomes CDC 1551 and H37Rv. We then used EpiMatrix, a proprietary pattern-matching algorithm, to do a preliminary analysis of these proteins for regions that contained a high number of class II MHC binding motif matches. The use of bioinformatics tools reduced the number of potential epitopes to be screened to 5% of the 1.3 million overlapping peptides. Peripheral blood mononuclear cells (PBMC) were obtained from healthy, asymptomatic tuberculin skin test-positive donors. Of the 17 highest-ranking peptide candidates that could be synthesized for this preliminary in vitro evaluation, 15 (88%) stimulated IFN-γ response, and eight (47%) stimulated lymphocyte proliferation in vitro. IFN-γ ELISpot assays were therefore a more sensitive test for T cell response to these peptides than were proliferation assays. One highly promiscuous epitope (MT2281-26-J, WRRRPLSSALLSFGLLLGGLPL) induced IFN-γ secretion in PBMC from 11 of 25 Mtb immune subjects (44%). Overall, 15 epitopes, and MT2281-26-J in particular, are candidates for inclusion in a multi-epitope TB vaccine. These findings support the systematic application of bioinformatics tools to whole genomes when used in combination with in vitro methods for screening and confirming epitopes.